Nitrile hydratase

ABSTRACT

Improving wild nitrile hydratase enables the provision of a protein which has nitrile hydratase activity and which has further improved heat resistance, amide compound resistance and high temperature accumulation properties. Use protein (A) or (B), (A) being a protein characterised by having nitrile hydratase activity and by including an amino acid sequence in which a specific amino acid residue in an amino acid sequence in wild nitrile hydratase has been substituted by another amino acid residue, and (B) being a protein characterised by having nitrile hydratase activity and by including an amino acid sequence in which one or several amino acid residues in the amino acid sequence of protein (A), other than the abovementioned specific amino acid residue, is deleted, substituted and/or added.

TECHNICAL FIELD

The present invention relates to an improved (mutant) nitrile hydrataseand its production method. The present invention also relates to genomicDNA that encodes the enzyme, a recombinant vector containing the genomicDNA, a transformant containing the recombinant vector, and a method forproducing an amide compound.

DESCRIPTION OF BACKGROUND ART

In recent years, a nitrile hydratase was found, which is an enzymehaving nitrile hydrolysis activity that catalyses the hydration of anitrile group to its corresponding amide group. Also, methods aredisclosed to produce corresponding amide compounds from nitrilecompounds using the enzyme or a microbial cell or the like containingthe enzyme. Compared with conventional chemical synthetic methods, suchmethods are known by a high conversion rate or selectivity rate from anitrile compound to a corresponding amide compound.

Examples of microorganisms that produce a nitrile hydratase are thegenus Corynebacterium, genus Pseudomonas, genus Rhodococcus, genusRhizobium, genus Klebsiella, genus Pseudonocardia and the like. Amongthose, Rhodococcus rhodochrous J1 strain has been used for industrialproduction of acrylamides, and its usefulness has been verified.Furthermore, a gene encoding a nitrile hydratase produced by the J1strain has been identified (see patent publication 1).

Meanwhile, introduction of a mutation into a nitrile hydratase has beenattempted not only as a way to use a nitrile hydratase isolated fromnaturally existing microorganisms or the gene of such a nitrilehydratase, but also as a way to change the activity, substratespecificity, Vmax, Km, heat stability, stability in a substrate,stability in a subsequent product and the like of a nitrile hydratase(see patent publication 2). Nitrile hydratase genes with improved heatresistance and amide-compound resistance have been produced by theinventors of the present invention (patent publications 3 and 4).

However, considering production costs such as the cost of catalysts whenproducing amide compounds, it is useful to develop a nitrile hydratasewith further improved heat resistance and amide-compound resistance, andenhanced capability of reacting at a high temperature. Thus, obtainingenzymes with such improved properties is highly desired for the purposeof reducing the amount of enzymes needed for reactions, loweringproduction costs and the like.

PRIOR ART PUBLICATION Patent Publication

-   Patent publication 1: Japanese patent publication 3162091-   Patent publication 2: International publication pamphlet    WO2004/056990-   Patent publication 3: International publication pamphlet    WO2005/116206-   Patent publication 4: Japanese laid-open patent publication    2007-143409

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The objective of the present invention is to improve a nitrile hydrataseso as to provide a protein having an improved nitrile hydratase activitywith enhanced heat resistance, amide-compound resistance andhigh-temperature accumulation properties. Another objective of thepresent invention is to provide a nitrile hydratase collected fromgenomic DNA encoding the protein, a recombinant vector containing thegenomic DNA, a transformant containing the recombinant vector, and aculture of the transformant, as well as a method for producing such anitrile hydratase. Yet another objective of the present invention is toprovide a method for producing an amide compound using the culture orthe processed product of the culture.

Solutions to the Problems

The inventors of the present invention have conducted extensive studiesto solve the above problems. As a result, in the amino acid sequence ofa wild-type nitrile hydratase, the inventors have found that a proteinin which a specific amino-acid residue is substituted with anotheramino-acid residue exhibits a nitrile hydratase activity with enhancedheat resistance, amide-compound resistance and high-temperatureaccumulation properties. Accordingly, the present invention isaccomplished.

Namely, the present invention is as follows.

(1) Protein (A) or (B) below:(A) A protein having nitrile hydratase activity and characterized by thefollowing: in the amino-acid sequence of a wild-type nitrile hydratase,amino-acid residues described in (a), (b), (c), (d) and (e) below arcsubstituted with other amino-acid residues, and at least one amino-acidresidue selected from among (f)˜(q) below is substituted with anotheramino-acid residue:

(a) the amino-acid residue at position 167 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(b) the amino-acid residue at position 219 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(c) the amino-acid residue at position 57 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(d) the amino-acid residue at position 114 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(e) the amino-acid residue at position 107 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(f) the amino-acid residue at position 218 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(g) the amino-acid residue at position 190 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(h) the amino-acid residue at position 168 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(i) the amino-acid residue at position 144 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(j) the amino-acid residue at position 133 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(k) the amino-acid residue at position 112 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(l) the amino-acid residue at position 105 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(m) the amino-acid residue at position 95 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(n) the amino-acid residue at position 17 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(o) the amino-acid residue at position 15 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(p) the amino-acid residue at position 67 counted downstream from thefurthermost downstream side C residue of the amino-acid sequenceC(S/T)LCSC that forms a binding site with a prosthetic molecule in theamino-acid sequence of the α subunit; and

(q) the amino-acid residue at position 17 counted downstream from thefurthermost downstream side C residue of the amino-acid sequenceC(S/T)LCSC that forms a binding site with a prosthetic molecule in theamino-acid sequence of the α subunit.

(B) A protein having nitrile hydratase activity and characterized by thefollowing: one or multiple amino-acid residues are deleted, substitutedand/or added in the amino-acid sequence of protein (A) excluding theamino-acid residues after the above substitution.(2) DNA encoding the protein described in (1);(3) a recombinant vector containing the genomic DNA described in (2);(4) a transformant containing the recombinant vector described in (3);(5) a nitrile hydratase collected from a culture obtained by incubatingthe transformant described in (4);(6) a method for producing a nitrile hydratase characterized bycollecting a nitrile hydratase from the culture obtained by incubatingthe transformant described in (4); and(7) a method for producing an amide compound characterized by a nitrilecompound being brought into contact with the protein described in (1) orwith a culture or a processed product of the culture obtained byincubating the transformant described in (4).

Effects of the Invention

According to the present invention, a novel improved (mutant) nitrilehydratase is obtained with enhanced heat resistance, amide-compoundresistance and high-temperature accumulation properties. The improvednitrile hydratase with further enhanced heat resistance, amide-compoundresistance and high-temperature accumulation properties is very usefulbecause it can produce amide compounds at a high yield.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing the structure of plasmid (pER855A);

FIG. 2-1 shows amino-acid sequences for β subunits of wild-type nitrilehydratases derived from various microorganisms;

FIG. 2-2 shows amino-acid sequences for β subunits of wild-type nitrilehydratases derived from various microorganisms (continued from FIG.2-1);

FIG. 3-1 shows amino-acid sequences for α subunits of wild-type nitritehydratases derived from various microorganisms; and

FIG. 3-2 shows amino-acid sequences for α subunits of wild-type nitrilehydratases derived from various microorganisms (continued from FIG.3-1).

MODE TO CARRY OUT THE INVENTION

In the following, the present invention is described in detail.

In the present application, unless otherwise specified, “upstream” and“upstream side” mean “N-terminal side” of an amino-acid sequence and “5′terminal side” of a base sequence.

Also, “downstream” and “downstream side” mean “C-terminal side” of anamino-acid sequence, and “3′ terminal side” of a base sequence.

1. Improved Nitrile Hydratase (a) Wild-Type Nitrile Hydratase

An improved nitrile hydratase of the present invention is derived from awild-type nitrile hydratase but is not limited to any specific wildtype. Here, a “wild-type nitrile hydratase” indicates: a nitrilehydratase isolated from living organisms found in nature (microorganismssuch as soil bacteria, for example); the amino-acid sequence forming theenzyme and the base sequence of the gene encoding the enzyme are notartificially deleted, inserted, or substituted by other amino acids orbases; and the nitrile hydratase retains the naturally existing originalproperties.

In addition to a nitrile hydratase identified to be derived from a knownmicroorganism, a nitrile hydratase identified by a DNA sequence forwhich the specific origin is not known may also be included in the above“wild-type nitrile hydratase.”

A “wild-type nitrile hydratase” has a higher-order structure formed withα and β subunit domains, and contains a non-heme iron atom or anon-corrin cobalt atom as a prosthetic molecule. Such a nitrilehydratase is identified and referred to as an iron-containing nitrilehydratase or a cobalt-containing nitrile hydratase.

An example of the iron-containing nitrile hydratase is derived fromRhodococcus N-771 strain. The conformation of such an iron-containingnitrile hydratase has been identified by X-ray crystal structuralanalysis. The enzyme is bonded with non-heme iron by four amino-acidresidues in a cysteine cluster (SEQ ID NO: 61) forming the active siteof the α subunit.

As for the cobalt-containing nitrile hydratase, examples are thosederived from Rhodococcus rhodochrous J1 strain (hereinafter may bereferred to as “J1 strain”) or derived from Pseudonocardia thermophila.A cobalt-containing nitrile hydratase derived from the J1 strain isbonded with a cobalt atom by a site identified as a cysteine cluster(SEQ ID NO: 62) that forms the active site of the α subunit. In thecysteine cluster of a cobalt-containing nitrile hydratase derived fromPseudonocardia thermophila, cysteine (Cys) at position 4 from theupstream side (N-terminal side) of the cysteine cluster derived from theJ1 strain is cysteine sulfinic acid (Csi), and cysteine (Cys) atposition 6 counted from the furthermost downstream side (C-terminalside) is cysteine sulfenic acid (Cse).

As described above, a prosthetic molecule is bonded with a siteidentified as cysteine clusters “C(S/T)LCSC” (SEQ ID NO: 61 and 62) inan α subunit. Examples of a nitrile hydratase containing a binding sitewith such a prosthetic molecule are those that have amino-acid sequencesand are encoded by genomic sequences derived from the following:Rhodococcus rhodochrous J1 (FERM BP-1478), Rhodococcus rhodochrous M8(SU 1731814), Rhodococcus rhodochrous M33 (VKM Ac-1515D), Rhodococcusrhodochrous ATCC 39484 (JP 2001-292772), Bacillus smithii (JPH9-248188), Pseudonocardia thermophila (JP H9-275978), or Geobacillusthermoglucosidasius.

FIGS. 3-1 and 3-2 show the alignments of amino-acid sequences (inone-letter code) in α-subunits of wild-type nitrile hydratases derivedfrom various microorganisms. From the top in FIGS. 3-1 and 3-2respectively, numbers 4 and 49˜60 of amino-acid sequences are shown.

On the other hand, β-subunits are thought to be attributed to structuralstability. FIGS. 2-1 and 2-2 show the alignments of amino-acid sequences(in one-letter code) for β subunits of wild-type nitrile hydratasesderived from various microorganisms. From the top in FIGS. 2-1 and 2-2respectively, numbers 2 and 35˜48 of amino-acid sequences are shown.

(b) Improved Nitrile Hydratase

The present invention relates to an improved (mutant) nitrile hydrataseformed by substituting amino acids of a wild-type nitrile hydratase.Amino-acid sequences of wild-type nitrile hydratases to be substitutedare made in public in NCBI databases such as GenBank(http://www.ncbi.nlm.nih.gov/) and the like.

For example, in the α subunit derived from Rhodococcus rhodochrous J1strain (FERM BP-1478), its amino-acid sequence is shown as SEQ ID NO: 4,and its base sequence is shown as SEQ ID NO: 3. Also, in the β subunit,its amino-acid sequence is shown as SEQ ID NO: 2, its base sequence isshown as SEQ ID NO: 1 and its accession number is “P21220”. In addition,the accession number of the α subunit for Rhodococcus rhodochrous M8 (SU1731814) is “ATT 79340” and the accession number for the β subunit is“AAT 79339.” Moreover, the accession number for the α subunit derivedfrom Pseudonocardia thermophila JCM 3095 is “1 IRE A” and the accessionnumber for the β subunit is “1 IRE B.”

Furthermore, also included in the scope of the present invention is animproved nitrile hydratase having nitrile hydratase activity andcharacterized as follows: in an amino-acid sequence in which a specificamino-acid residue is substituted, at least one amino-acid residue (forexample, approximately 1˜10, preferably 1˜5, amino-acid residues,excluding the amino-acid residue after substitution) is deleted,substituted and/or added in the amino-acid sequence.

An “improved nitrile hydratase” of the present invention is a proteinhaving nitrile hydratase activity and characterized as follows: in theamino-acid sequence of a wild-type nitrile hydratase, amino-acidresidues identified as (a), (b), (c), (d) and (e) are substituted withother amino-acid residues, and at least one amino-acid residue selectedfrom a group of (f)˜(q) below is substituted with another amino-acidresidue. Amino-acid residues identified as (d), (e), (f), (g), (h), (i),(j), (k), (l), (m), (n) (o), (p) and (q) are each preferred to be anamino-acid residue in the amino-acid sequence of a wild-type nitrilehydratase derived from a bacterium that belongs to the Rhodococcusrhodocrous species among various wild-type nitrile hydratases.

(a) an amino-acid residue at position 167 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of a β subunit;

(b) the amino-acid residue at position 219 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(c) the amino-acid residue at position 57 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(d) the amino-acid residue at position 114 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(e) the amino-acid residue at position 107 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(f) the amino-acid residue at position 218 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(g) the amino-acid residue at position 190 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(h) the amino-acid residue at position 168 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(i) the amino-acid residue at position 144 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(j) the amino-acid residue at position 133 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(k) the amino-acid residue at position 112 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(l) the amino-acid residue at position 105 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(m) the amino-acid residue at position 95 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(n) the amino-acid residue at position 17 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(o) the amino-acid residue at position 15 counted downstream from theN-terminal amino-acid residue in the amino-acid sequence of the βsubunit;

(p) the amino-acid residue at position 67 counted downstream from thefurthermost downstream side C residue of the amino-acid sequenceC(S/T)LCSC that forms a binding site with a prosthetic molecule in theamino-acid sequence of the α subunit; and

(q) the amino-acid residue at position 17 counted downstream from thefurthermost downstream side C residue of the amino-acid sequenceC(S/T)LCSC that forms a binding site with a prosthetic molecule in theamino-acid sequence of the α subunit.

Furthermore, as for the improved nitrile hydratase of the presentinvention, it is preferred that the amino-acid residues to besubstituted in the above examples of protein be the amino-acid residueslisted below.

(1) amino-acid residues identified in (a)˜(e) and (n) above.(2) amino-acid residues identified in (a)˜(e) and (n) above, and atleast one selected from a group of (g)˜(j), (1), (m), (O) and (q);(3) amino-acid residues identified in (a)˜(e), (n), (i) and (p) above.(4) amino-acid residues identified in (a)˜(e), (n), (p) and (f) above.(5) amino-acid residues identified in (a)˜(e), (n), (i) and (p) abovealong with at least one amino-acid residue selected from a group of (f),(k) and (m) above.

An example of the improved nitrile hydratase of the present invention ispreferred to be an enzymatic protein containing the followingamino-acids in the amino-acid sequence of a wild-type nitrile hydratasederived from the J1 strain, and having nitrile hydratase activity: anamino-acid residue (asparagine) at position 167 counted from theN-terminal side in the amino-acid sequence of the β subunit; anamino-acid residue (valine) at position 219 counted from the N-terminalside in the amino-acid sequence of the β subunit; an amino-acid residue(serine) at position 57 counted from the N-terminal side in theamino-acid sequence of the β subunit; an amino-acid residue (lysine) atposition 114 counted from the N-terminal side in the amino-acid sequenceof the β subunit; an amino-acid residue (threonine) at position 107counted from the N-terminal side in the amino-acid sequence of the βsubunit; and an amino-acid residue (proline) at position 17 counted fromthe N-terminal side in the amino-acid sequence of the β subunit.Examples of code abbreviations to show such amino-acid substitutions are“Nβ167S, Vβ219A, Sβ57R, Kβ114Y, Tβ107K, Pβ17D” and the like.

Amino acids are coded by a single letter of the alphabet. The letter onthe left of the numeral that shows the number of amino-acid residuesexisting between the terminal and the substituted position (26, forexample) is a one-letter code before substitution, and the letter on theright is a one-letter code of the amino acid after substitution.

More specifically, when the amino-acid sequence of the β subunit shownas SEQ ID NO: 2 is written as “Nβ167S,” it shows the amino-acidsubstitution performed in the improved nitrile hydratase; namely, in theamino-acid sequence of the β subunit (SEQ ID NO: 2), asparagine (N) atposition 167 counted from the N-terminal amino-acid residue (includingthe N-terminal amino-acid residue itself) is substituted with serine(S).

Here, “α↓” means the substituted position is located downstream from thefurthermost downstream C residue in the CTLCSC site (on the C-terminalside excluding the C residue itself).

Moreover, as for an improved nitrile hydratase of the present invention,amino-acid residues to be substituted in the protein above are preferredto be those shown in Table 1.

TABLE 1 substitution substitution site number of amino acid specificmode of amino-acid substitution 1 (f) Cβ218H 2 (g) Gβ190H 3 (h) Kβ168R 4(i) Lβ144R, Lβ144S 5 (j) Lβ133N, Lβ133R 6 (k) Sβ112T 7 (l) Rβ105W 8 (m)Kβ95V 9 (n) Pβ17D Pβ17H Pβ17G Pβ17S 10 (o) Pβ15S 11 (p) Gα ↓ 67L 12 (q)Eα ↓ 17S 13 (a), (b), (c), (d), (e) and Nβ167S, Vβ219A, Sβ57M, Kβ114Y,Tβ107K, Pβ17D (n) 14 (a), (b), (c), (d), (e) and Nβ167S, Vβ219A, Sβ57M,Kβ114Y, Tβ107K, Pβ17H (n) 15 (a), (b), (c), (d), (e) and Nβ167S, Vβ219A,Sβ57M, Kβ114Y, Tβ107K, Pβ17G (n) 16 (a), (b), (c), (d), (e) and Nβ167S,Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S (n) 17 (a), (b), (c), (d), (e), (n)Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, and (l) Rβ105W 18 (a),(b), (c), (d), (e), (n) Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S,and (i) Lβ144S 19 (a), (b), (c), (d), (e), (n) Nβ167S, Vβ219A, Sβ57M,Kβ114Y, Tβ107K, Pβ17S, and (h) Kβ168R 20 (a), (b), (c), (d), (e), (n)Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, and (q) Eα ↓ 17S 21 (a),(b), (c), (d), (e), (n) Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S,and (g) Gβ190H 22 (a), (b), (c), (d), (e), (n) Nβ167S, Vβ219A, Sβ57M,Kβ114Y, Tβ107K, Pβ17S, and (m) Kβ95V 23 (a), (b), (c), (d), (e), (n)Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, and (j) Lβ133N 24 (a),(b), (c), (d), (e), (n) Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S,and (j) Lβ133R 25 (a), (b), (c), (d), (e), (n) Nβ167S, Vβ219A, Sβ57M,Kβ114Y, Tβ107K, Pβ17S, and (o) Pβ15S 26 (a), (b), (c), (d), (e), (n),Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, (i) and (p) Lβ144S, Gα ↓67L 27 (a), (b), (c), (d), (e), (n), Nβ167S, Vβ219A, Sβ57M, Kβ114Y,Tβ107K, Pβ17S, (i) and (p) Lβ144S, Gα ↓ 67V 28 (a), (b), (c), (d), (e),(n), Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, (p) and (f) Gα ↓ 67L,Cβ218H 29 (a), (b), (c), (d), (e), (n), Nβ167S, Vβ219A, Sβ57M, Kβ114Y,Tβ107K, Fβ17S, (i), (p) and (m) Lβ144S, Gα ↓ 67L, Kβ95V 30 (a), (b),(c), (d), (e), (n), Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, (i),(p) and (k) Lβ144S, Gα ↓ 67L, Sβ112T 31 (a), (b), (c), (d), (e), (n),Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, (i), (p) and (f) Lβ144S,Gα ↓ 67L, Cβ218H

Among those, improved nitrile hydratases in which amino-acid residuesare substituted as shown in substitution numbers 13˜29 are preferred,and especially preferred are those in substitution numbers 26˜29.

As for base substitutions to cause amino-acid substitutions as above,substitutions shown in Table 2 below are preferred.

TABLE 2 amino-acid substitution base substitution Cβ218H Codon “TGC” atpositions 652~654 downstream (3′ terminal side) from the first A of basesequence ATG (positions 1~3 in SEQ ID NO: 1) is substituted with CAT orCAC. Especially preferred to be substituted is T at position 652downstream with C, and G at position 653 downstream with A (TGC→CAC).Gβ190H Codon “GGC” at positions 568~570 downstream (3′ terminal side)from the first A of base sequence ATG (positions 1~3 in SEQ ID NO: 1) issubstituted with CAT or CAC. Especially preferred to be substituted is Gat position 568 downstream with C, and G at position 569 downstream withA (GGC→CAC). Kβ168R Codon “AAG” at positions 502~504 downstream (3′terminal side) from the first A of base sequence ATG (positions 1~3 inSEQ ID NO: 1) is substituted with CGT, CGC, CGA, CGG, AGA or AGG.Especially preferred to be substituted is A at position 503 downstreamwith G (AAG→AGG). Lβ144R Codon “CGT” at positions 430~432 downstream (3′terminal side) from the first A of base sequence ATG (positions 1~3 inSEQ ID NO: 1) is substituted with CGT, CGC, CGA, CGG, AGA or AGG.Especially preferred to be substituted is C at position 258 downstreamwith A, and T at position 257 downstream with G (CTC→AGG). Lβ144S Codon“CGT” at positions 430~432 downstream (3′ terminal side) from the firstA of base sequence ATG (positions 1~3 in SEQ ID NO: 1) is substitutedwith TCT, TCC, TCA, TCG, AGT or AGC. Especially preferred to besubstituted is C at position 430 downstream with A, T at position 431downstream with G, and G at position 432 downstream with C (CTC→AGC).Lβ133N Codon “CTA” at positions 397~399 downstream (3′ terminal side)from the first A of base sequence ATG (positions 1~3 in SEQ ID NO: 1) issubstituted with AAT or AAC. Especially preferred to be substituted is Cat position 397 downstream with A, T at position 398 with A, and A atposition 399 downstream with C (CTA→AAC). Lβ133R Codon “CTA” atpositions 397~399 downstream (3′ terminal side) from the first A of basesequence ATG (positions 1~3 in SEQ ID NO: 1) is substituted with CGT,CGC, CGA, CGG, AGA or AGG. Especially preferred to be substituted is Tat position 398 downstream with G, and A at position 399 downstream withC (CTA→CGC). Sβ112T Codon “TCG” at positions 334~336 downstream (3′terminal side) from the first A of base sequence ATG (positions 1~3 inSEQ ID NO: 1) is substituted with ACT, ACC, ACA or ACG. Especiallypreferred to be substituted is T at position 334 downstream with A, andG at position 336 downstream with C (TCG→ACC). Rβ105W Codon “CGG” atpositions 313~315 downstream (3′ terminal side) from the first A of basesequence ATG (positions 1~3 in SEQ ID NO: 1) is substituted with TGG.Especially preferred to be substituted is C at position 313 downstreamwith T (CGG→TGG). Kβ95V Codon “AAG” at positions 283~285 downstream (3′terminal side) from the first A of base sequence ATG (positions 1~3 inSEQ ID NO: 1) is substituted with CTT, CTC, GTA or GTG. Especiallypreferred to be substituted is A at position 283 downstream with G, andA at position 284 downstream with T (AAG→GTG). Pβ17D Codon “CCC” atpositions 49~51 downstream (3′ terminal side) from the first A of basesequence ATG (positions 1~3 in SEQ ID NO: 1) is substituted with GAT orGAC. Especially preferred to be substituted is C at position 49downstream with G, and C at position 50 downstream with A (CCC→GAC).Pβ17H Codon “CCC” at positions 49~51 downstream (3′ terminal side) fromthe first A of base sequence ATG (positions 1~3 in SEQ ID NO: 1) issubstituted with CAT or CAC. Especially preferred to be substituted is Cat position 50 downstream with A (CCC→CAC). Pβ17G Codon “CCC” atpositions 49~51 downstream (3′ terminal side) from the first A of basesequence ATG (positions 1~3 in SEQ ID NO: 1) is substituted with GGT,GGC, GGA or GGG. Especially preferred to be substituted is C at position49 downstream with G, C at position 50 downstream with G, C at position50 downstream with G, and C at position 51 downstream with G (CCC→GGG).Pβ17S Codon “CCC” at positions 49~51 downstream (3′ terminal side) fromthe first A of base sequence ATG (positions 1~3 in SEQ ID NO: 1) issubstituted with TCT, TCC, TCA, TCG, AGT or AGC. Especially preferred tobe substituted is C at position 49 downstream with A, and C at position50 downstream with G (CCC→AGC). Pβ15S Codon “CCG” at positions 43~45downstream (3′ terminal side) from the first A of base sequence ATG(positions 1~3 in SEQ ID NO: 1) is substituted with TCT, TCC, TCA, TCG,AGT or AGC. Especially preferred to be substituted is C at position 49downstream with A, and C at position 50 downstream with G (CCG→AGC).Gα↓67L Codon “GGT” at positions 199~201 downstream (toward 3′ terminal)from the last C of base sequence TGCACTCTGTGTTCGTGC (positions 304~321in SEQ ID NO: 3) is substituted with TTA, TTG, CTT, CTC, CTA or CTG.Especially preferred to be substituted is G at position 199 downstreamwith T, G at position 200 downstream with T, and T at position 201downstream with G (GGT→TTG). Gα↓67V Codon “GGT” at positions 199~201downstream (toward 3′ terminal) from the last C of base sequenceTGCACTCTGTGTTCGTGC (positions 304~321 in SEQ ID NO: 3) is substitutedwith GTA, GTC, GTG or GTT. Especially preferred to be substituted is Gat position 200 downstream with T, and T at position 201 downstream withG (GGT→GTG). Gα↓17S Codon “GAG” at positions 49~51 downstream (toward 3′terminal) from the last C of base sequence TGCACTCTGTGTTCGTGC (positions304~321 in SEQ ID NO: 3) is substituted with TCT, TCC, TCA, TCG, AGT orAGC. Especially preferred to be substituted is G at position 49downstream with A, and A at position 50 downstream with G, G at position51 downstream with C (GAG→AGC).

In the alignment of the β-subunit of a nitrile hydratase derived fromthe J1 strain shown as SEQ ID NO: 2, the positions of amino acids to besubstituted in the present invention include 167, 219, 57, 114, 107,218, 190, 168, 144, 133, 112, 105, 95, 17 and 15. For example, if it isa Pseudonocardia thermophila, positions of amino acids to be substitutedin the amino-acid sequence are 164, 216, 57, 114, 107, 215, 187, 165,141, 129, 108, 102, 92, 17 and 15. Moreover, as for positions of aminoacids to be substituted in the present invention, positions 124 and 174of the α-subunit of the J1 strain of nitrile hydratase identified as SEQID NO: 4 and positions 130 and 180 of Pseudonocardia thermophila arealso included.

Aligning the amino-acid sequence is not limited to any specific method.For example, a genomic sequence analysis software such as GENTXY (NipponGenetics Co, Ltd.), DNASIS (Hitachi Solutions, Ltd.), or a free softwareCLUSTALW or BLAST may be used. FIGS. 2-1, 2-2, 3-1 and 3-2 show thealignment results obtained by using version 7, GENETXY (default setting,Nippon Genetics Co., Ltd.)

The improved nitrile hydratase activity of the present invention showsenhanced heat resistance, amide-compound resistance and high-temperatureaccumulation properties compared with a wild-type nitrile hydrataseactivity retaining the naturally existing original characteristics.

Here, “nitrile hydratase activity” means an enzymatic activity tocatalyze the hydration for converting a nitrile compound to acorresponding amide compound (RCN+H₂O→RCONH₂). Determining the activityis conducted by bringing a nitrile compound as a substrate into contactwith a nitrile hydratase for conversion to a corresponding amidecompound and by measuring the resultant amide compound. Any nitrilecompound may be used as a substrate as long as nitrile hydratase reactswith such the compound, but acrylonitrile is preferred.

Reaction conditions are a substrate concentration of 2.5%, reactiontemperature of 10° C. to 30° C. and duration of 10˜30 minutes. Theenzymatic reactions are terminated by adding phosphoric acid. Then,using HPLC (high-performance liquid chromatography), the producedacrylamide is analyzed to measure the amount of the amide compound.

In addition, the presence of nitrile hydratase activity is simplyexamined by activity staining. For example, if anthranilonitrile is usedas a substrate, since anthranilamide converted by a nitrile hydrataseyields fluorescent, nitrile hydratase activity is easily detected athigh sensitivity (reference: Antonie Van Leeuwenhoek, 80(2): 169-183,2001).

“Improved heat resistance” means that the remaining activity of aheat-treated improved strain is at least 10% higher than the remainingactivity of the comparative example treated the same way. The method forheat treatment is to supply a liquid culture, or collected and washedbacterial-cell culture, in a container and to place the container in aheating device such as a water bath or incubator so as to maintain thetemperature for a predetermined duration. At that time, to enhance thestability of the enzyme, a nitrile compound or an amide compound may beadded for such heat treatment. For treatment conditions, the temperatureand duration are preferred to be set in such a way that the activity ofthe comparative example becomes no more than 50% of that prior to theheat treatment. In particular, heat treatment is preferred to beperformed in a temperature range of 50° C. to 70° C. for 5 minutes to 60minutes. Remaining activity means the ratio of the amount of an amidecompound produced by heat-treated bacterial cells for activitymeasurement to the amount of an amide compound produced by the sameamount of untreated bacterial cells for activity measurement. Untreatedbacterial cells are those in a liquid culture or collected and washedbacterial-cell culture, which are refrigerated at a temperature of 4° C.The comparative example in the present invention is a transformant intowhich pER855A is introduced. When a nitrile hydratase shows a remainingactivity at least 10% higher than that of the comparative strain, theheat resistance of the nitrile hydratase is confirmed to be improved.

“Amino-compound resistance” means that a nitrile hydratase can maintainits activity in the presence of an amide compound. A culturedtransformant containing an improved nitrile hydratase, or an improvednitrile hydratase isolated from the transformant, is analyzed in thepresence of an amide compound such as acrylamide (at a highconcentration of 30˜50%, for example) to examine the consumption amountor consumption rate of a nitrile compound such as acrylonitrile for asubstrate. When the consumption amount or consumption rate exceeds 1.01times that of the comparative example, the nitrile hydratase isconfirmed to be resistant to amide compounds.

“High-temperature accumulation properties” means that a nitrilehydratase is capable of producing acrylamide at a high concentrationexceeding 35% at a reaction temperature of 20° C. or higher. Enzymaticreactions of a cultured transformant containing an improved nitrilehydratase, or an improved nitrile hydratase isolated from thetransformant, are continued by adding acrylonitrile, and then theconcentration of a produced acrylamide is analyzed. Acrylonitrile may beadded while the acrylonitrile content in the reaction mixture iscontrolled, or may be added sequentially to continue reactions. Hightemperature in the present application means a reaction temperature of20° C. or higher. When the concentration of produced acrylamide exceedsthat of the comparative example, the high-temperature accumulationproperties of the improved nitrile hydratase is evaluated to beenhanced.

An example of amide compounds is represented by general formula (1)below:

R—CONH₂  (1)

(Here, “R” is a straight-chain or branched alkyl or alkenyl group having1˜10 carbon atoms with an optional substituent, a cycloalkyl group orallyl group having 3˜18 carbon atoms with an optional substituent, or asaturated or unsaturated heterocyclic group with an optionalsubstituent.) Especially, an acrylamide is preferred to have “CH2=CH—”as “R” in the formula.

The above improved nitrile hydratase is obtained by substituting aminoacids of a wild-type nitrile hydratase. For example, the improvednitrile hydratase is obtained by modifying the amino-acid sequence (SEQID NO: 2 and/or 4) of a nitrile hydratase derived from Rhodococcusrhodochrous J1 strain and by selecting a nitrile hydratase with enhancedheat resistance and/or amide-compound resistance.

Rhodococcus rhodochrous J1 strain is internationally registered as FERMBP-1478 at the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (Central 6,1-1-1 Higashi, Tsukuba, Ibaraki), deposited Sep. 18, 1987.

In nitrile hydratases other than the J1 strain, enzymes with a highlyhomologous amino-acid sequence are thought to acquire enhanced heatresistance and/or amide-compound resistance through the mutation above.

As for such strains, Bacillus smithii (JP H09-248188), Pseudonocardiathermophila (JP H09-275978), Geobacillus thermoglucosidasius and thelike are listed. Especially preferred are Rhodococcus rhodochrous M8 (SU1731814) and Rhodococcus rhodochrous M33 (VKM Ac-1515D), in whichamino-acid homology is 90% or higher (high homology enzymes are listed).Rhodococcus rhodochrous M33 (VKM Ac-1515D) is selected as a result ofthe natural mutation of the M8 strain (SU 1731814) above, and is capableof constitutive expression of nitrile hydratase, but the amino-acidsequence or genomic sequence of the nitrile hydratase itself is notmodified (U.S. Pat. No. 5,827,699).

Methods for conducting amino-acid substitution on a wild-type nitrilehydratase are as follows: a bacterium having nitrile hydratase activityis brought into contact for reactions with chemicals such ashydroxylamine or nitrous acid as a mutation source; UV rays areirradiated to induce mutation; error-prone PCR or site-directedmutagenesis is employed to introduce a mutation at random into the genethat encodes a nitrile hydratase; and the like.

(b-1) Method for Introducing Random Mutation

To study functions and characteristics of proteins using a mutant,random mutagenesis is known. Random mutagenesis is a method to introducea random mutation to the gene encoding a specific protein so that amutant is produced. In random mutagenesis by PCR, stringency conditionsare set low during DNA amplification to introduce mutation in a basesequence (error-prone PCR).

In such an error-prone PCR method, a mutation is introduced randomlyinto any position of the entire DNA site to be amplified. Then, byexamining the function of the obtained mutant into which a mutation wasintroduced randomly, information of amino acids or domains important forthe specific functions of a protein is obtained.

For a nitrile hydratase as the template of error-prone PCR, the nitrilehydratase gene derived from a wild type strain or DNA obtained as anamplified product by error-prone PCR are used.

As reaction conditions for error-prone PCR, for example, a compositionratio of any one, two or three among dNTP (dGTP, dCTP, dATP or dTTP) inthe reaction mix is reduced relative to another dNTP. In so setting,during the DNA synthesis, at a position that requires a dNTP whose ratiois reduced, another dNTP is more likely to be used by error, and thatmay lead to mutation. In addition, other preferred reaction conditionsare a composition in which the amount of MgCl₂ and/or MnCl₂ in thereaction mix is increased.

(b-2) Improved Nitrile Hydratase Derived from Rhodococcus rhodochrous J1Strain and its Gene

An improved nitrile hydratase of the present invention includes the geneencoding a protein into which mutations shown in Table 1 are introduced.

Based on a wild-type nitrile hydratase gene, DNA that encodes such animproved nitrile hydratase is produced by site-directed mutagenesismethods described in Molecular Cloning, A Laboratory Manual, 2ndedition, published by Cold Spring Harbor Laboratory Press (1989),Current Protocols in Molecular Biology, John Wiley and Sons (1987-1997)and the like. To introduce a mutation into DNA by well-known methodssuch as the Kunkel method or the Gapped Duplex method, mutagenesis kitsapplying site-directed mutagenesis methods such as follows are used:QuickChange™ XL Site-Directed Mutagenesis Kit (made by Stratagene),GeneTailor™ Site-Directed Mutagenesis System (made by InvitrogenCorporation), TaKaRa Site-Directed Mutagenesis System (Mutan-K,Mutan-Super Express Km and the like, made by Takara Bio Inc.) and thelike.

Furthermore, a gene related to the present invention includes DNA whichis hybridized under stringent conditions with a DNA made up of a basesequence complementary to the base sequence of the gene of the presentinvention, and which encodes a protein having nitrile hydrataseactivity.

Such an improved nitrile hydratase gene is obtained by introducing amutation into a wild-type gene as described above. Alternatively, usingthe genomic sequence or its complementary sequence or a DNA fragment asa probe, improved nitrile hydratase gene may also be obtained from cDNAlibraries and genomic libraries by employing well-known hybridizationmethods such as colony hybridization, plaque hybridization, Southernblot or the like. Libraries constructed by a well-known method may beused, or commercially available cDNA libraries and genomic libraries mayalso be used.

“Stringent conditions” are those for washing after hybridization; a saltconcentration of 300˜2000 mM, a temperature of 40˜75° C., preferably asalt concentration of 600˜900 mM, and a temperature of 65° C. Forexample, conditions 2×SSC at 50° C. may be employed. In addition to sucha salt concentration of the buffer, temperature and the like, a personskilled in the art may set conditions for obtaining DNA that encodes anitrile hydratase of the present invention by adding various conditionssuch as probe concentration, probe length and reaction time.

For detailed procedures for hybridization, Molecular Cloning, ALaboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press(1989)) and the like may be referred to. DNA to be hybridized includesDNA or its fragment, containing a base sequence which is at least 40%,preferably 60%, more preferably 90% or greater, homologous to thegenomic DNA of the present invention.

An amino acid (amino acid after substitution) that substitutes aspecific amino acid residue in the amino-acid sequence of a wild-typenitrile hydratase is not limited to any specific type, and may beselected properly as long as the polypeptide (protein) that includes theamino acid after substitution exhibits nitrile hydratase activity.

(c) Recombinant Vector, Transformant

It is necessary for a nitrile hydratase gene to be put into a vector sothat nitrile hydratase is expressed in the host organism to betransformed. Examples of such vectors are plasmid DNA, bacteriophageDNA, retrotransposon DNA, artificial chromosome DNA and the like.

In addition, a host to be used in the present invention is not limitedto any specific type as long as the target nitrile hydratase isexpressed after the recombinant vector is introduced into the host.Examples are bacteria such as E. coli and Bacillus subtilis, yeasts,animal cells, insect cells, plant cells and the like. When E. coli isused as a host, an expression vector with high expression efficiency,such as expression vector pkk 233-2 with a trc promoter (made byAmersham Biosciences Corp.), pTrc 99A (made by Amersham BiosciencesCorp.) or the like, is preferred.

In addition to a nitrile hydratase gene, a vector may be coupled with apromoter, terminator, enhancer, splicing signal, poly A addition signal,selection marker, ribosome binding sequence (SD sequence) or the like.Examples of selection markers are a kanamycin resistance gene,dihydrofolate reductase gene, ampicillin resistance gene, neomycinresistance gene and the like.

When a bacterium is used as a host, Escherichia coli may be used, forexample, and a Rhodococcus strain such as Rhodococcus rhodochrous ATCC12674, Rhodococcus rhodochrous ATCC 17895 and Rhodococcus rhodochrousATCC 19140 may also be used. Those ATCC strains are obtained from theAmerican type culture collection.

When E. coli is used as a host for producing a transformant to express anitrile hydratase, since most of the expressed nitrile hydratases areformed as inclusion bodies and are insoluble, a transformant with lowcatalytic activity is obtained. On the other hand, if a Rhodococcusstrain is used as a host, nitrile hydratase is present in the solublefraction and a transformant with high activity is obtained. Thosetransformants may be selected based on purposes. However, when animproved enzyme is selected under stringent conditions, a transformantwith high activity derived from a Rhodococcus strain is preferred.

Introducing a recombinant vector into a bacterium is not limited to anyspecific method as long as DNA is introduced into the bacterium. Forexample, a method using calcium ions, electroporation or the like may beemployed.

When yeast is used as a host, examples are Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia pastoris and the like. As a method forintroducing a recombinant vector into yeast, it is not limitedspecifically as long as DNA is introduced into the yeast. For example,an electroporation method, spheroplast method, lithium acetate method orthe like may be employed.

When animal cells are used as a host, monkey cells COS-7, Vero, CHOcells, mouse L cells, rat GH3 cells, human FL cells or the like may beemployed. As a method for introducing a recombinant vector into ananimal cell, for example, an electroporation method, calcium phosphatemethod, lipofection method or the like may be used.

When insect cells are used as a host, Sf9 cells, Sf21 cells and the likemay be used. As a method for introducing a recombinant vector into aninsect cell, for example, a calcium phosphate method, lipofectionmethod, electroporation method or the like may be used.

When plant cells are used as a host, tobacco BY-2 cells or the like maybe used. However, that is not the only option. A method for introducinga recombinant vector into a plant cell, for example, an Agrobacteriummethod, particle gun method, PEG method, electroporation method or thelike may be used.

(d) Method for Producing Culture and Improved Nitrile Hydratase

An improved nitrile hydratase of the present invention is obtained byincubating the above transformant and by collecting from the obtainedculture.

The present invention also relates to a method for producing an improvednitrile hydratase, and the method is characterized by collecting animproved nitrile hydratase from the culture above.

In the present invention, “culture” means culture supernatant, culturedcells, cultured bacterial cells, cell homogenates or bacterial-cellhomogenates. Incubation of a transformant of the present invention isconducted by a generally used method for incubating a host. The targetnitrile hydratase is accumulated in the culture.

As for a culture to incubate a transformant of the present invention,any natural or synthetic culture medium is used as long as it contains acarbon source, a nitrogen source, inorganic salts or the like for thehost bacteria to assimilate, and incubation of a transformant isperformed efficiently. Examples of a carbon source are carbohydratessuch as glucose, galactose, fructose, sucrose, raffinose and starch;organic acids such as acetic acid and propionic acid; alcohols such asethanol and propanol; and the like. Examples of a nitrogen source areinorganic acids such as ammonia, ammonium chloride, ammonium sulfate,ammonium acetate and ammonium phosphate; ammonium salts of organicacids; and other nitrogen-containing compounds.

In addition, peptone, yeast extract, meat extract, corn steep liquor,various amino acids or the like may also be used. Examples of mineralsare monopotassium phosphate, potassium dihydrogenphosphate, magnesiumphosphate, magnesium sulfate, sodium chloride, ferrous sulfate,manganese sulfate, zinc sulfate, copper sulfate, calcium carbonate andthe like. Also, if necessary, a defoaming agent may be used to preventfoaming during the incubation process. Moreover, cobalt ions or ironions as prosthetic molecules of a nitrile hydratase, or nitriles andamides as an inducer of the enzyme, may also be added to the culture.

Incubation may be conducted by adding selective pressure to prevent thevector and the target gene from being eliminated. Namely, if a selectionmarker is a drug-resistant gene, a corresponding chemical agent may beadded; or if a selection marker is an auxotrophic complementary gene,corresponding nutrition factors may be removed.

Also, if a selection marker has a genetic assimilation trait, anequivalent assimilation factor may be added as a sole factor ifnecessary. For example, when E. coli transformed by a vector containingan ampicillin-resistant gene is incubated, ampicillin may be added asneeded during the incubation process.

When incubating a transformant transformed by an expression vectorcontaining an inducible promoter, such an inducer may be added to theculture if necessary. For example, when incubating a transformanttransformed by an expression vector with a promoter inducible withisopropyl-β-D-thiogalactopyranoside (IPTG), IPTG or the like may beadded to the culture. Likewise, when incubating a transformanttransformed by an expression vector with a trp promoter inducible withindoleacetic acid (IAA), IAA or the like may be added to the culture.

Incubation conditions of a transformant are not limited specifically aslong as the productivity of the target nitrile hydratase and growth ofthe host are not prohibited. Generally, conditions are preferred to be10° C.˜40° C., more preferably 20° C.˜37° C., for 5˜100 hours. The pHvalue is adjusted using inorganic or organic acid, alkaline solution orthe like. If it is an E. coli, the pH is adjusted to be 6˜9.

As for incubation methods, solid-state culture, static culture, shakingculture, aeration-agitation culture and the like may be used. When an E.coli transformant is incubated, it is especially preferred to useshaking culture or aeration-agitation culture (jar fermentation) underaerobic conditions.

When incubated in culture conditions above, the improved nitrilehydratase of the present invention is accumulated at a high yield in theabove culture medium, namely, at least in any of culture supernatant,cell culture, bacterial-cell culture, cell homogenates or bacterial-cellhomogenates.

When an improved nitrile hydratase is incubated and produced in a cellor bacterial cell, the target nitrile hydratase is collected byhomogenizing the cell or bacterial cell. Cells or bacterial cells arehomogenized by high-pressure treatment using a French press orhomogenizer, supersonic treatment, grinding treatment using glass beadsor the like, enzyme treatment using lysozyme, cellulose, pectinase andthe like, freezing and thawing treatment, hypotonic solution treatment,bacteriolysis induction treatment by phage, and so on.

After the homogenization process, residues of the cell homogenates orbacterial-cell homogenates (including insoluble fractions of the cellextract) are removed if necessary. To remove residues, centrifugal orfiltration methods are employed. To increase the efficiency of removingresidues, a coagulant or filter aid may be used. The supernatantobtained after the removal of residues is soluble fractions of the cellextract, which are used as a crudely purified improved nitrile hydratasesolution.

Also, when an improved nitrile hydratase is produced in cells orbacterial cells, it is an option to collect the cells or bacterial cellsthemselves by a centrifuge or membrane filtration and to use withouthomogenizing them.

When an improved nitrile hydratase is produced outside the cells orbacterial cells, the culture may be used as is, or the cells orbacterial cells are removed using a centrifugal or filtration method.Then, the improved nitrile hydratase is collected from the culture bybeing extracted through ammonium sulfate precipitation, if necessary.Furthermore, dialysis or various chromatography techniques (gelfiltration, ion exchange chromatography, affinity chromatography, etc.)may be used to isolate and purify the nitrile hydratase.

To check the production yield of a nitrile hydratase obtained byincubating a transformant is not limited to using any specific method,but SDS-PAGE (polyacrylamide gel electrophoresis), nitrile hydrataseactivity measurements or the like may be used to determine the yield perculture, per wet or dry weight in a bacterial cell, or per crudeenzymatic protein. SDS-PAGE may be conducted by a method well known by aperson skilled in the art. Also, the activity described above may beapplied to nitrile hydratase activity.

Without using any living cells, an improved nitrile hydratase of thepresent invention may be produced using a cell-free protein synthesissystem.

In a cell-free protein synthesis system, a protein is produced in anartificial vessel such as a test tube using a cell extract. A cell-freeprotein synthesis system used in the present application includes acell-free transcription system that synthesizes RNA using DNA as atemplate.

In such a case, an organism corresponding to the above host is theorganism from which the cell extract is derived. Here, for the cellextract, extracts of eukaryotic or prokaryotic origin, such as theextract from wheat germ, E. coli and the like, may be used. Such cellextracts may be concentrated or not.

The cell extract is obtained by ultrafiltration, dialysis, polyethyleneglycol (PEG) precipitation or the like. In the present invention, acommercially available kit may also be used for cell-free proteinsynthesis. Examples of such a kit are a reagent kit PROTEIOS™ (Toyobo),TNT™ system (PromegaKK), a synthesizer PG-Mate™ (Toyobo), RTS (RocheDiagnostics) and the like.

An improved nitrile hydratase obtained by cell-free protein synthesis asdescribed above is also purified by properly selecting a chromatographytype.

2. Method for Producing Amide Compound

The improved nitrile hydratase obtained above is used as an enzymaticcatalyst when producing material. For example, an amide compound isproduced by bringing a nitrile compound into contact with the improvednitrile hydratase. Then, the amide compound produced upon contact iscollected. Accordingly, an amide compound is produced.

The isolated and purified nitrile hydratase as described above is usedas an enzymatic catalyst. In addition, a gene is introduced so as toexpress an improved nitrile hydratase in a proper host as describedabove and the culture after the host is incubated or the processedproducts of the culture may also be used. Processed products are, forexample, incubated cells immobilized with acrylamide gel or the like,those processed by glutaraldehyde, those supported by inorganic carrierssuch as alumina, silica, zeolite, diatomaceous earth and the like.

Here, “contact” means that an improved nitrile hydratase and a nitrilecompound are present in the same reaction system or incubation system:for example, an isolated and purified improved nitrile hydratase and anitrile compound are mixed; a nitrile compound is added into aincubation vessel of a cell to express an improved nitrile hydratasegene; cells are incubated in the presence of a nitrile compound; a cellextract is mixed with a nitrile compound; and so on.

A nitrile compound as a substrate is selected by considering thesubstrate specificity of the enzyme, stability of the enzyme in thesubstrate and the like. As for a nitrile compound, acrylonitrile ispreferred. The reaction method and the method for collecting an amidecompound after the completion of reactions are properly selecteddepending on the characteristics of the substrate and the enzymaticcatalyst.

The enzymatic catalyst is preferred to be recycled as long as itsactivity is not deactivated. From the viewpoint of preventingdeactivation and of recycling ease, the enzymatic catalyst is preferredto be used as a processed product.

EXAMPLES

In the following, examples of the present invention are described indetail. However, present invention is not limited to those.

Example 1 Obtaining Improved Nitrile Hydratase Gene and EvaluationThereof (1) (1) Construction of Mutant Gene Library

The plasmid used as a template was plasmid pER855A (FIG. 1) modifiedfrom plasmid pER855 (see JP 2010-172295) as follows: in the β subunit ofthe amino-acid sequence (SEQ ID NO: 2), the amino-acid residuepositioned at 167 downstream from the N-terminal amino-acid residue wasmutated from asparagine (N) to serine (S); the amino-acid residuepositioned at 219 downstream from the N-terminal amino-acid residueabove was mutated from valine (V) to alanine (A); the amino-acid residuepositioned at 57 downstream from the N-terminal amino-acid residue abovewas mutated from serine (S) to methionine (M); the amino-acid residuepositioned at 114 downstream from the N-terminal amino-acid residueabove was mutated from lysine (K) to tyrosine (Y); and the amino-acidresidue positioned at 107 downstream from the N-terminal amino-acidresidue above was mutated from threonine (T) to lysine (K).

Plasmid pSJ034 used as a vector is capable of expressing a nitrilehydratase in a Rhodococcus strain, and was prepared from pSJ023 using amethod described in JP H10-337185. Here, pSJ023 is a transformant “R.rhodochrous ATCC 12674/pSJ023,” and is internationally registered asFERM BP-6232 at the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (Central 6,1-1-1 Higashi, Tsukuba, Ibaraki), deposited Mar. 4, 1997.

First, a mutation was introduced into the nitrile hydratase gene usingthe following method.

<Composition of PCR Reaction Mixture>

sterile water 20 μL pER855A (1 ng/mL)  1 μL Forward primer (10 mM)  2 μLReverse primer (10 mM)  2 μL PrimeSTAR MAX (2x) 25 μL 50 μL

<PCR Reaction Conditions>

(98° C. for 10 sec, 55° C. for 5 sec, 72° C. for 90 sec)×30 cycles

<Primers>Primer for Saturation Mutagenesis at β17

β17RM-F: ggatacggaccggtcNNStatcagaaggacgag (SEQ ID NO: 5)β17RM-R: ctcgtccttctgataSNNgaccggtccgtatcc (SEQ ID NO: 6)

<Reaction Conditions>

(94° C. for 30 sec, 65° C. for 30 sec, 72° C. for 3 min)×30 cycles

After the completion of PCR, 5 μL of the reaction mixture was providedfor 0.7% agarose gel electrophoresis, an amplified fragment of 11 kb wasconfirmed, and 1 μL of DpnI (provided with the kit) was added to the PCRreaction mixture, which was then reacted at 37° C. for an hour.Accordingly, the plasmid template was removed. After that, the reactionmixture was purified using Wizard SV Gel and PCR Clean-Up System(Promega KK), and transformation was introduced into JM109 by thepurified PCR reaction product. A few thousand obtained colonies werecollected from the plate, and plasmid DNA was extracted using QIAprepSpin Miniprep Kit (Qiagen) to construct a mutant-gene library.

(2) Producing Rhodococcus Transformant

The cells of Rhodococcus rhodochrous strain ATCC 12674 at a logarithmicgrowth phase were collected by a centrifugal separator, washed withice-cooled sterile water three times and suspended in the sterile water.Then, 1 μL of plasmid prepared in (1) above and 10 μL of thebacterial-cell suspension were mixed and ice-cooled. The plasmid DNA andthe bacterial-cell suspension were supplied into a cuvette, and electricpulse treatment was conducted at 2.0 KV and 200Ω using anelectroporation device, Gene Pulser II (Bio-Rad Laboratories, Inc.).

The cuvette with the mixture processed by electric pulses was let standfor 10 minutes under ice-cold conditions, and a heat-shock treatment wasconducted at 37° C. for 10 minutes. Then, 500 μL of an MYK culturemedium (0.5% polypeptone, 0.3% Bacto yeast extract, 0.3% Bacto maltextract, 0.2% K₂HPO₄, 0.2% KH₂PO₄) was added and let stand at 30° C. for5 hours, and the strain was applied onto an MYK agar medium containing50 μg/mL kanamycin. The colony obtained after incubating at 30° C. for 3days was used as a transformant. In the same manner, transformantpER855A was prepared as a comparative strain.

(3) Amide Treatment on Rhodococcus Transformant

The Rhodococcus transformant containing a nitrile hydratase geneobtained in (2) above and ATCC 12674/pER855A as a comparative strainwere used for screening. In a 96-hole deep-well plate, 1 mL each of aGGPK culture medium (1.5% glucose, 1% sodium glutamate, 0.1% yeastextract, 0.05% K₂HPO₄, 0.05% KRA) O₄, 0.05% MgSO₄.7H₂O, 1% CoCl₂, 0.1%urea, 50 μg/mL kanamycin, pH at 7.2) was supplied. In each culturemedium, the above strain was inoculated, and liquid culture was carriedout at 30° C. for 3 days.

Next, 30 μL of the liquid culture obtained above was dispensed in a96-hole plate and the culture medium was removed by centrifugation.Lastly, 40 μL of 50% acrylamide solution was added to suspend thebacteria. The transformant suspended in a high-concentration acrylamidesolution was put in an incubator to completely deactivate thecomparative strain through heat treatment conducted at 50° C. for 30minutes. The remaining nitrile hydratase activity was measured asfollows.

First, after the acrylamide treatment, the transformant was washed witha 50 mM phosphate buffer (pH 7.0) and the activity was measured by thefollowing method. The washed transformant and 50 mM phosphate buffer (pH7.0) were supplied to a test tube and preincubated at 30° C. for 10minutes, and an equivalent volume of a 5% acrylonitrile solution (pH7.0) was added and reacted for 10 minutes. Then, one tenth volume of 1 Mphosphoric acid was added to terminate the reaction. Next, thetransformant was removed from the terminated reaction mixture by acentrifuge, and the mixture was diluted to a proper concentration foranalysis by HPLC (WAKOSIL 5C8 (Wako Pure Chemical Industries) 250 mmlong, 10% acetonitrile containing 5 mM phosphoric acid, flow rate ofmobile phase at 1 mL/min, wavelength of a UV absorption detector 260nm). Using untreated cells for which acrylamide treatment was notconducted, activity was measured for comparison. Then, based on theobtained activity values, the activity remaining after the completion ofacrylamide treatment was determined.

Among hundreds of transformants each containing a nitrile hydratase geneinto which a mutation was introduced as above, four strains of mutantenzymes showing resistance to a high concentration of acrylamide wereselected as shown in Table 3.

TABLE 3 mutant strain No. name of plasmid 1 pFR003 2 pFR004 3 pFR005 4pFR006

(4) Confirming Base Sequence

To confirm the base sequence of a nitrile hydratase gene, plasmid wasrecovered from the selected strains. The Rhodococcus transformant wasinoculated into 10 mL of an MYK culture medium (0.5% polypeptone, 0.3%Bacto yeast extract, 0.3% malt extract, 1% glucose, 50 μg/mL kanamycin)and incubated for 24 hours, and a 20% sterile glycine solution was addedto make the final concentration of 2%, which was further incubated foranother 24 hours. Then, the bacterial cells were recovered by acentrifuge, washed with TES buffer (10 mM Tris-HCl (pH8)-10 mM NaCl-1 mMEDTA), suspended in 2 mL of 50 mM Tris-HCl (pH8)-12.5% sucrose-100 mMNaCl-1 mg/mL lysozyme, and shaken at 37° C. for 3 hours. Then, 0.4 mL of10% SDS was added and the mixture was shaken gently for an hour at roomtemperature, to which 2.1 mL of 5 M sodium acetate buffer (pH 5.2) wasadded and let stand in ice for an hour. Next, the mixture wascentrifuged for an hour at 10,000×g at 4° C. to obtain a supernatant, towhich a 5-times volume of ethanol was added and let stand at −20° C. for30 minutes. Then, the mixture was centrifuged at 10,000×g for 20minutes. The precipitant was washed with 10 mL of 70% ethanol anddissolved in 100 μL of a TE buffer. Accordingly, a DNA solution wasobtained.

Next, the sequence including a nitrile hydratase was amplified by a PCRmethod.

<Composition of PCR Reaction Mixture>

template plasmid 1 μL 10× PCR buffer (made by NEB) 10 μL  primer NH-19(50 μM) 1 μL primer NH-20 (50 μM) 1 μL 2.5 mM dNTPmix 8 μL sterile water79 μL  Taq DNA polymerase (made by NEB) 1 μL

<primers> (SEQ ID NO: 7) NH-19: GCCTCTAGATATCGCCATTCCGTTGCCGG(SEQ ID NO: 8) NH-20: ACCCTGCAGGCTCGGCGCACCGGATGCCCAC

<Reaction Conditions>

(94° C. for 30 sec, 65° C. for 30 sec, 72° C. for 3 min)×30 cycles

After completion of PCR, 5 μL of the reaction mixture was subjected to0.7% agarose gel electrophoresis to detect a PCR amplified fragment of2.5 kb. After Exo-SAP treatment (Amersham Pharmacia Biotech) on the PCRreaction mixture, samples for alignment analysis were prepared by acycle sequencing method, and were analyzed using CEQ-2000XL (BeckmanCoulter, Inc). The results are shown in Table 4.

TABLE 4 name of plasmid mutation site pSJ 034 no mutation site pER 855ANβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K (template) pFR 003 Nβ167S, Vβ219A,Sβ57M, Kβ114Y, Tβ107K, Pβ17D pFR 004 Nβ167S, Vβ219A, Sβ57M, Kβ114Y,Tβ107K, Pβ17H pFR 005 Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17G pFR006 Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S

(5) Evaluation of Amide-Compound Resistance

The amide-compound resistance of the improved nitrile hydratasesobtained in (4) above was evaluated by the following method.

ATCC12674/pER855A and each transformant obtained in step (2) above werebrought into contact with 10 mL of an MYK culture medium (50 μg/mLkanamycin), and subjected to shaking culture at 30° C. for 2 days. Then,1% of each culture was inoculated into 100 mL of a GGPK culture medium(1.5% glucose, 1% sodium glutamate, 0.1% yeast extract, 0.05% K₂HPO₄,0.05% KH₂PO₄, 0.05% MgSO₄.7H₂O, 1% CoCl₂, 0.1% urea, 50 μg/mL kanamycin,pH 7.2), and subjected to shaking culture at 30° C. for 3 days. Then,bacterial cells were collected by centrifugation.

The enzyme activity of the obtained cultured cells was measured by thefollowing method: 0.2 mL of the bacterial cell mixture and 4.8 mL of a50 mM phosphate buffer (pH 7.0) were mixed, to which 5 mL of a 50 mMphosphate buffer (pH 7.0) containing 5.0% (w/v) acrylonitrile wasfurther added. Next, the mixture was reacted while being shaken at 10°C. for 10 minutes. Then, bacterial cells were filtered and the amount ofproduced acrylamide was measured using gas chromatography.

<Analysis Conditions>

-   analysis instrument: gas chromatograph GC-14B (Shimadzu Corporation)-   detector: FID (detection at 200° C.)-   column: 1 m glass column filled with PoraPak PS (column filler made    by Waters Corp.)-   column temperature: 190° C.

Nitrile hydratase activity was determined by conversion from the amountof acrylamide. Here, regarding nitrile hydratase activity, the amount ofenzyme to produce 1 μmol of acrylamide per 1 minute is set as 1 U.

Next, experiments were conducted by setting the composition of areaction mixture and reaction conditions below. Using the enzymeactivity measured in advance, each bacterial suspension used forreaction was properly diluted by 100 mM phosphate buffer (pH 7.0) sothat the amount of activity is set to be the same. ATCC 12674/pER855Awas used as a comparison strain.

<Composition of Reaction Mixture>

50% acrylamide solution: 94 g  acrylonitrile 4 g 1M phosphate buffer: 1g bacterial fluid (same unit (U) of enzymatic activity)

<Reaction Conditions>

reaction for 5 hours while being stirred (30° C.)

Before the start of reaction (0 hour) and 5 hours after the start ofreaction, 1 mL of the reaction mixture was taken for sampling, which wasthen filtered by a 0.45 μm filter. The obtained filtrate was put throughgas chromatography. The proportion of remaining acrylonitrile wasanalyzed. The results are shown in Table 5.

TABLE 5 proportion of acrylonitrile (%) consumption consumption before 5hrs after amount rate name of reaction reaction of acrylonitrile ofacrylonitrile plasmid starts (A) starts (B) (A − B) (%) pER 855A 4.010.81 3.20 100 (comparative example) pFR 003 4.01 0.70 3.31 103 pFR 0044.01 0.36 3.65 114 pFR 005 4.01 0.40 3.61 113 pFR 006 4.01 0.66 3.35 105

From the results above, in all the improved nitrile hydratases, theconsumption rates of acrylonitrile exceeded 103% relative to the resultof comparative example pER855A set at 100%. Thus, it is found thatnitrile hydratase activity was maintained in the presence ofhigh-concentration acrylamide and that resistance to acrylamide isenhanced in improved nitrile hydratase.

Example 2 Obtaining Improved Nitrile Hydratase Gene and EvaluationThereof (2)

(1) Introduction of Mutation into Nitrile Hydratase and SelectionThereof.

Using pFR005 obtained in Example 1 as a template, an attempt was made toobtain an improved nitrile hydratase having further enhanced acrylamideresistance. The same procedures were employed as in Example 1(introducing mutation, forming Rhodococcus transformant, amideprocessing of Rhodococcus transformant, confirming base sequence) exceptthat the primers were changed, and mutant enzymes shown in Table 6 wereobtained.

<primers> primer for saturation mutagenesis at β15 (SEQ ID NO: 9)β15RM-F: atgaccggatacggaNNSgtcccctatcagaag (SEQ ID NO: 10) β15RM-R:cttctgataggggacSNNtccgtatccggtcatprimer for saturation mutagenesis at β95 (SEQ ID NO: 11) β95RM-F:accgaagaagagcgaNNScaccgtgtgcaagag (SEQ ID NO: 12) β95RM-R:ctcttgcacacggtgSNNtcgctcttcttcggtprimer for saturation mutagenesis at β105 (SEQ ID NO: 13) β105RM-F:GAGATCCTTGAGGGTNNSTACACGGACAGG (SEQ ID NO: 14) β105RM-R:CCTGTCCGTGTASNNACCCTCAAGGATCTC primer for saturation mutagenesis at β133(SEQ ID NO: 15) β133RM-F: cacgagccccactccNNSgcgcttccaggagcg(SEQ ID NO: 16) β133RM-R: cgctcctggaagcgcSNNggagtggggctcgtgsaturation mutagenesis primer at β144 (SEQ ID NO: 17) β144RM-F:ggagccgagtttctctNNSggtgacaagatc (SEQ ID NO: 18) β144RM-R:gatcttgtcaccSNNagagaaactcggctccprimer for saturation mutagenesis at β168 (SEQ ID NO: 19) β168RM-F:cgaaatatgtgcggagcNNSatcggggaaatcg (SEQ ID NO: 20) β168RM-R:cgatttccccgatSNNgctccgcacatatttcgprimer for saturation mutagenesis atβ190 (SEQ ID NO: 21) β190RM-F:gagcagctccgccggcctcNNSgacgatcctcg (SEQ ID NO: 22) β90RM-R:cgaggatcgtcSNNgaggccggcggagctgctcprimer for saturation mutagenesis at α124 (SEQ ID NO: 23) α124RM-F:gtacaagagcatgNNStaccggtcccgagtgg (SEQ ID NO: 24) α124RM-R:ccactcgggaccggtaSNNcatgctcttgtac

TABLE 6 name of plasmid mutation site pFR005 (template) Nβ167S, Vβ219A,Sβ57M, Kβ114Y, Tβ107K, Pβ17G pFR102 Nβ167S, Vβ219A, Sβ57M, Kβ114Y,Tβ107K, Pβ17S, Rβ105W pFR108A Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K,Pβ17S, Lβ144S pFR109 Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S,Kβ168R pFR112 Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, Eα↓17SpFR116 Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, Gβ190H pFR119Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, Kβ95V pFR120 Nβ167S,Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, Lβ133N pFR121 Nβ167S, Vβ219A,Sβ57M, Kβ114Y, Tβ107K, Pβ17S, Lβ133R pFR122 Nβ167S, Vβ219A, Sβ57M,Kβ114Y, Tβ107K, Pβ17S, Pβ15S

(2) Performance Evaluation

The performance of obtained improved nitrile hydratases was evaluated bythe same method as in (5) of Example 1.

TABLE 7 proportion of acrylonitrile (%) consumption before 5 hrs afteramount consumption reaction reaction of acrylonitrile rate of name ofplasmid starts (A) starts (B) (A − B) acrylonitrile pER855A 4.01 0.813.20 100 (comp example) pFR102 4.01 0.15 3.86 121 pFR108A 4.01 0.15 3.86121 pFR109 4.01 0.25 3.76 118 pFR112 4.01 0.17 3.84 120 pFR116 4.01 0.153.86 121 pFR119 4.01 0.25 3.76 117 pFR120 4.01 0.15 3.86 121 pFR121 4.010.05 3.96 124 pFR122 4.01 0.21 3.80 119

From the results above, in all the improved nitrile hydratases, theconsumption rates of acrylonitrile exceeded 117% relative to the resultof comparative example pER855A set at 100%. Thus, it is found thatnitrile hydratase activity was maintained in the presence ofhigh-concentration acrylamide and that resistance to acrylamide isenhanced in improved nitrile hydratase.

Example 3 Obtaining Improved Nitrile Hydratase Gene and EvaluationThereof (3)

(1) Introduction of Mutation into Nitrile Hydratase and SelectionThereof.

Using pFR108A obtained in Example 2 as a template, an attempt was madeto obtain an improved nitrile hydratase having further enhancedacrylamide resistance. The same procedures were employed as in Example 1(introducing mutation, forming Rhodococcus transformant, amideprocessing of Rhodococcus transformant, confirming base sequence) exceptthat the primers were changed, and mutant enzymes shown in Table 8 wereobtained. Selection of transformant containing an improved nitrilehydratase was conducted using the same method as in Example 1 exceptthat heat treatment was conducted at 55° C. for 60 minutes.

<primers> primer for saturation mutagenesis at α174 (SEQ ID NO: 25)α174RM-F: gccggcaccgacNNStggtccgaggag (SEQ ID NO: 26) α174RM-R:ctcctcggaccaSNNgtcggtgccggc

TABLE 8 name of plasmid mutation site pFR108A Nβ167S, Vβ219A, Sβ57M,Kβ114Y, Tβ107K, (template) Pβ17S, Lβ144S pFR211 Nβ167S, Vβ219A, Sβ57M,Kβ114Y, Tβ107K, Pβ17S, Lβ144S, Gα↓67L pFR212 Nβ167S, Vβ219A, Sβ57M,Kβ114Y, Tβ107K, Pβ17S, Lβ144S, Gα↓67V

(2) Performance Evaluation

The performance of obtained improved nitrile hydratase was evaluated bythe same method as in (5) of Example 1.

TABLE 9 proportion of acrylonitrile (%) consumption before 5 hrs afteramount consumption reaction reaction of acrylonitrile rate of name ofplasmid starts (A) starts (B) (A − B) acrylonitrile pER855A 4.01 0.813.20 1.00 (comp example) pFR211 4.01 0.05 3.96 1.24 pFR212 4.01 0.053.96 1.24

From the results above, in all the improved nitrile hydratases, theconsumption rates of acrylonitrile exceeded 124% relative to the resultof comparative example pER855A set at 100%. Thus, it is found thatnitrile hydratase activity was maintained in the presence ofhigh-concentration acrylamide and that resistance to acrylamide isenhanced in improved nitrile hydratase.

Example 4 (1) Introduction of Mutation into Nitrile Hydratase andSelection Thereof

Using pFR211 obtained in Example 2 as a template, an attempt was made toobtain an improved nitrile hydratase having further enhanced acrylamideresistance. The same procedures were used as in Example 3 (introducingmutation, forming Rhodococcus transformant, amide processing ofRhodococcus transformant, confirming base sequence) except that theprimers were changed, and mutant enzymes shown in Table 10 wereobtained.

<primers> primer for saturation mutagenesis at β95 (SEQ ID NO: 27)β95RM-F: accgaagaagagcgaNNScaccgtgtgcaagag (SEQ ID NO: 28) β95RM-R:ctcttgcacacggtgSNNtcgctcttcttcggtprimer for saturation mutagenesis at β112 (SEQ ID NO: 29) β112RM-F:GACAGGAAGCCGNNSCGGAAGTTCGATCCG (SEQ ID NO: 30) β112RM-R:CGGATCGAACTTCCGSNNCGGCTTCCTGTC primer for saturation mutagenesis at β218(SEQ ID NO: 31) β218RM-F: gggaaagacgtagtgNNSgccgatctctgggaa(SEQ ID NO: 32) β218RM-R: ttcccagagatcggcSNNcactacgtattccc

TABLE 10 name of plasmid mutation site pFR303 Nβ167S, Vβ219A, Sβ57M,Kβ114Y, Tβ107K, Pβ17S, Lβ144S, Gα↓67L, Kβ95V pFR304 Nβ167S, Vβ219A,Sβ57M, Kβ114Y, Tβ107K, Pβ17S, Lβ144S, Gα↓67L, Sβ112T pFR306 Nβ167S,Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, Lβ144S, Gα↓67L, Cβ218H

(2) Performance Evaluation

The performance of obtained improved nitrile hydratases was evaluated bythe same method as in (5) of Example 1. The results are shown in Table11

TABLE 11 proportion of acrylonitrile (%) consumption before 3 hrs afteramount consumption reaction reaction of acrylonitrile rate of name ofplasmid starts (A) starts (B) (A − B) acrylonitrile pER855A 4.01 0.813.20 1.00 (comp example) pFR303 4.01 0.00 4.01 1.25 pFR304 4.01 0.004.01 1.25 pFR306 4.01 0.00 4.01 1.25

From the results above, in all the improved nitrile hydratases, theconsumption rates of acrylonitrile exceeded 125% relative to the resultof comparative example pER855A set at 100%. Thus, it is found thatnitrile hydratase activity was maintained in the presence ofhigh-concentration acrylamide and that resistance to acrylamide isenhanced in improved nitrile hydratase.

Example 5 (1) Producing pFR306A

Using pFR306 obtained in Example 4 as a template, an improved nitrilehydratase was produced by substituting Lβ144S with a wild-type aminoacid. A Rhodococcus transformant was produced using the same method asin Example 1 and the primers below.

<primers> mutation at β144 is returned to a wild type (SEQ ID NO: 33)F-Sβ144L-F: TTCTCTCTCGGTGACAAGATCAAAGTG (SEQ ID NO: 34) F-Sβ144L-R:GTCACCGAGAGAGAAACTCGGCTCCGC

TABLE 12 name of plasmid mutation site pFR306A Nβ167S, Vβ219A, Sβ57M,Kβ114Y, Tβ107K, Pβ17S, Gα↓67L, Cβ218H

(2) Evaluation of Heat Resistivity

Performance of improved nitrile hydratases obtained in the presentinvention was evaluated as follows.

Transformants containing mutant nitrile hydratase genes shown in Table13 were incubated by the method in (5) of Example 1 to evaluate heatresistivity. After the cultures were diluted properly by a 50 mMphosphate buffer and heated in a 70° C. water bath for 10 minutes,remaining nitrile hydratase activity was determined by the methoddescribed in (5) of Example 1. For comparison, untreated bacteriasamples were prepared by not heating the cultures but keeping them at 4°C., and their respective remaining activity was determined.

TABLE 13 name of plasmid mutation site pER855A Nβ167S, Vβ219A, Sβ57M,Kβ114Y, Tβ107K (comp example) pFR005 Nβ167S, Vβ219A, Sβ57M, Kβ114Y,Tβ107K, Pβ17S pFR108A Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S,Lβ144S pFR211 Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, Lβ144S,Gα↓67L pFR303 Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S, Lβ144S,Gα↓67L, Kβ95V pFR304 Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K, Pβ17S,Lβ144S, Gα↓67L, Sβ112T pFR306 Nβ167S, Vβ219A, Sβ57M, Kβ114Y, Tβ107K,Pβ17S, Lβ144S, Gα↓67L, Cβ218H pFR306A Nβ167S, Vβ219A, Sβ57M, Kβ114Y,Tβ107K, Pβ17S, Gα↓67L, Cβ218H

When the remaining activity of comparative example pER855A was set as 1(11%), the remaining activity of each of all the improved nitrilehydratases was at least 3 times (30%) as great. Thus, the heatresistivity of improved nitrile hydratases was found to be enhanced.

Example 6

Using transformants obtained in Example 5, their acrylamide accumulationproperties during high-temperature reactions was evaluated.

In a plastic tube with a lid, 10 mL of a 50 mM phosphate buffer and atransformant were added and preincubated for 10 minutes while the tubewas shaken in a 40° C. water bath. Next, 1 mL of acrylonitrile was addedto each reaction mixture and a reaction was started. After the start ofthe reaction, 1 mL each of acrylonitrile at predetermined timing (20minutes, 40 minutes, 1 hour, 1½ hours, 2 hours) was added to continuethe reaction. The reaction mixture 3 hours after the start of thereaction was filtered and the acrylamide concentration of the filtratewas determined by gas chromatography.

As a result of the experiment, 32% of the acrylamide was accumulated incomparative example pER855A while over 40% of the acrylamide wasaccumulated in each improved nitrile hydratase. Accordingly, improvednitrile hydratases were found to have enhanced high-temperatureaccumulation properties.

INDUSTRIAL APPLICABILITY

An improved nitrile hydratase is provided by the present invention. Inthe improved nitrile hydratase of the present invention, heatresistance, amide-compound resistance and high-temperature accumulationproperties are enhanced. Thus, using the improved nitrile hydratase ofthe present invention, amide compounds are produced efficiently fromnitrile compounds.

DETAILS OF SEQUENCE LISTING

-   SEQ ID NO:1 base sequence in β subunit of a nitrile hydratase    derived from J1 strain-   SEQ ID NO:2 amino-acid sequence in β subunit of a nitrile hydratase    derived from J1 strain-   SEQ ID NO:3 base sequence in α subunit of a nitrile hydratase    derived from J1 strain-   SEQ ID NO:4 amino-acid sequence in α subunit of a nitrile hydratase    derived from J1 strain-   SEQ ID NO:5 primer for saturation mutagenesis at β17-   SEQ ID NO:6 primer for saturation mutagenesis at β17-   SEQ ID NO:7 NH-19 primer-   SEQ ID NO:8 NH-20 primer-   SEQ ID NO:9 primer for saturation mutagenesis at β15-   SEQ ID NO:10 primer for saturation mutagenesis at β15-   SEQ ID NO:11 primer for saturation mutagenesis at β95-   SEQ ID NO:12 primer for saturation mutagenesis at β95-   SEQ ID NO:13 primer for saturation mutagenesis at β105-   SEQ ID NO:14 primer for saturation mutagenesis at β105-   SEQ ID NO:15 primer for saturation mutagenesis at β133-   SEQ ID NO:16 primer for saturation mutagenesis at β133-   SEQ ID NO:17 primer for saturation mutagenesis at β144-   SEQ ID NO:18 primer for saturation mutagenesis at β144-   SEQ ID NO:19 primer for saturation mutagenesis at β168-   SEQ ID NO:20 primer for saturation mutagenesis at β168-   SEQ ID NO:21 primer for saturation mutagenesis at β190-   SEQ ID NO:22 primer for saturation mutagenesis at β190-   SEQ ID NO:23 primer for saturation mutagenesis at α124-   SEQ ID NO:24 primer for saturation mutagenesis at α124-   SEQ ID NO:25 primer for saturation mutagenesis at α174-   SEQ ID NO:26 primer for saturation mutagenesis at α174-   SEQ ID NO:27 primer for saturation mutagenesis at β95-   SEQ ID NO:28 primer for saturation mutagenesis at β95-   SEQ ID NO:29 primer for saturation mutagenesis at β112-   SEQ ID NO:30 primer for saturation mutagenesis at β112-   SEQ ID NO:31 primer for saturation mutagenesis at β218-   SEQ ID NO:32 primer for saturation mutagenesis at β218-   SEQ ID NO:33 primer to return mutation at β144 to a wild type-   SEQ ID NO:34 primer to return mutation at β144 to a wild type-   SEQ ID NO:35 β subunit of Rhodococcus M8-   SEQ ID NO:36 β subunit of Rhodococcus ruber TH-   SEQ ID NO:37 β subunit of R. pyridinovorans MW3-   SEQ ID NO:38 β subunit of R. pyridinovorans S85-2-   SEQ ID NO:39 β subunit of R. pyridinovorans MS-38-   SEQ ID NO:40 β subunit of Nocardia sp. JBRs-   SEQ ID NO:41 β subunit of Nocardia sp. YS-2002-   SEQ ID NO:42 β subunit of R.rhodocrous ATCC39384-   SEQ ID NO:43 β subunit of uncultured bacterium SP1-   SEQ ID NO:44 β subunit of uncultured bacterium BD2-   SEQ ID NO:45 β subunit of Comamonas testosteroni-   SEQ ID NO:46 β subunit of G. theimoglucosidasius Q6-   SEQ ID NO:47 β subunit of P. thermophila JCM 3095-   SEQ ID NO:48 β subunit of R. rhodocrous Cr4-   SEQ ID NO:49 α subunit of Rhodococcus M8-   SEQ ID NO:50 α subunit of Rhodococcus ruber TH-   SEQ ID NO:51 α subunit of R. pyridinovorans MW3-   SEQ ID NO:52 α subunit of R. pyridinovorans S85-2-   SEQ ID NO:53 α subunit of Nocardia sp. JBRs-   SEQ ID NO:54 α subunit of Nocardia sp. YS-2002-   SEQ ID NO:55 α subunit of uncultured bacterium BD2-   SEQ ID NO:56 α subunit of uncultured bacterium SP1-   SEQ ID NO:57 α subunit of R. rhodocrous ATCC39484-   SEQ ID NO:58 α subunit of Sinorhizobium medicae WSM419-   SEQ ID NO:59 α subunit of P. thermophila JCM 3095-   SEQ ID NO:60 α subunit of R. rhodocrous Cr4-   SEQ ID NO:61 cysteine cluster in α subunit of iron-containing    nitrile hydratase derived from Rhodococcus N-771 strain-   SEQ ID NO:62 cysteine cluster in α subunit of cobalt-containing    nitrile hydratase derived from J1 strain

1. A protein (A) or (B): (A) a protein, comprising nitrile hydrataseactivity, wherein in the amino-acid sequence of a wild-type nitrilehydratase, amino-acid residues at positions 167, 219, 57, 114, 107, and17 counted downstream from an N-terminal amino-acid residue in anamino-acid sequence of a β subunit are substituted with other amino-acidresidues, and an amino-acid residue at position 218, 190, 168, 144, 133,112, 105, 95, or 15 counted downstream from an N-terminal amino-acidresidue in an amino-acid sequence of a β subunit, an amino acid residueat position 67 or 17 counted downstream from a furthermost-downstreamside C residue of an amino-acid sequence C(S/T)LCSC that forms a bindingsite with a prosthetic molecule in an amino-acid sequence of an αsubunit, or any combination thereof is substituted with anotheramino-acid residue; or (B) a protein, comprising nitrile hydrataseactivity, wherein one or a plurality of amino-acid residues are deleted,substituted, added, or any combination thereof in an amino-acid sequenceof protein (A) in addition to amino-acid residue substitutions ofprotein (A).
 2. An isolated DNA encoding the protein according toclaim
 1. 3. An isolated DNA sequence that hybridizes with the isolatedDNA according to claim 2 under stringent conditions, and encoding aprotein with nitrile hydratase activity.
 4. A recombinant vectorcomprising the isolated DNA according to claim
 2. 5. A transformantcomprising the recombinant vector according to claim
 4. 6. A nitrilehydratase collected from a culture obtained by a process comprisingincubating the transformant according to claim
 5. 7. A method forproducing a nitrile hydratase, the method comprising: incubating thetransformant according to claim 5 and collecting a nitrile hydratasefrom the culture.
 8. A method for producing an amide compound, themethod comprising: bringing a nitrile compound into contact with theprotein according to claim
 1. 9. A method for producing an amidecompound, the method comprising: bringing a nitrile compound intocontact with a culture or a processed product of the culture obtained byincubating the transformant according to claim 5.